Alphaviruses are arthropod-borne viruses that cause summertime epidemics of rash and arthritis and encephalitis. Sindbis virus (SV) is an alphavirus that causes encephalomyelitis in mice by infecting and damaging neurons. The outcome of SV infection of neurons is determined by the virulence of the virus, by the ability of the cell to resist SV-induced apoptosis and by the immune response. We have shown that the E2 glycoprotein is a major viral determinant of virulence and that maturity is a major neuronal determinant of susceptibility to SV-induced apoptosis. Crucial interactions determining efficiency of infection and induction of apoptosis occur at the time of virus binding and entry. Binding to cells in culture occurs through interaction of the E2 glycoprotein with heparan sulfate-bearing proteoglycans, but other, as yet unidentified, virus receptors are present and can mediate pH-dependent fusion and entry. Fusion is dependent on the E1 glycoprotein and requires sphingomyelin in the cell membrane. During fusion, acidic sphingomyelinase is activated to degrade sphingomyelin and release ceramide initiating an apoptotic cascade leading to cell death. Viruses which vary in virulence differ in the efficiency with which these processes occur in neuronal cells. In this application we propose to define at a molecular level the viral determinants of virulence and how virulent viruses interact with target host cells through the following specific aims: (1) To identify the residues in the E2 glycoprotein that determine binding of SV to heparan sulfate and the role(s) of these residues in cell-specific binding. (2) To determine the role of SV binding to heparan sulfate in virus virulence. (3) To identify the coreceptor(s) for SV and the roles of the coreceptor(s) in cell tropism and virus entry. (4) To determine the role of cellular lipids in virus entry into neural and non neural cells. (5) To determine the role of cellular lipids and lipid-derived signalling molecules in SV-induced apoptosis.